The biochemical pathways of microorganisms can be classified as being part of either primary or secondary metabolism. The pathways of primary metabolism are involved in the catabolism of molecules for energy production or in the synthesis of the building blocks of the cells. Most of these processes are common for all microorganisms. The secondary metabolism is usually anabolic and leads to compounds with no obvious function for the cell.
Among secondary metabolites .beta.-lactam antibiotics are a large family produced in nature by microorganisms. The most important class of .beta.-lactam antibiotics both clinically and economically are the penicillins and the cephalosporins. Their biosynthesis occur via a complex pathway of enzymatic steps.
The unravelling of this pathway has been the subject of many studies during the last few decades. The first two steps in the biosynthetic pathways of the penicillin and the cephalosporin classes of .beta.-lactam antibiotics are identical. Thereafter the biosynthetic pathways to the penicillins and cephalosporins diverge.
The .beta.-lactam biosynthetic pathway
The pathway to the important penicillin species penicillin V is sketched below.
Aminoadipic acid+cysteine+valine.fwdarw.ACV-tripeptide.fwdarw.isopenicillin N.fwdarw.penicillin V
The first step is a condensation of L-.alpha.-aminoadipic acid (an intermediate in the lysine biosynthetic pathway in fungi or a degradation product of lysine in bacteria), L-cysteine and L-valine. In cephamycin-producing Actinomycetes, lysine (an amino acid belonging to the so-called aspartate family) is synthesized by the dihydrodipicolinate pathway, which does not include .alpha.-aminoadipic acid as an intermediate. In these organisms the precursor is formed by catabolism of lysine by the action of lysine-6-aminotransferase.
In the second step, ACV is oxidatively cyclized by removal of four hydrogen atoms to form the bicyclic penam nucleus (a .beta.-lactam fused to a thiazolidine ring) of isopenicillin N which is present in all penicillins. From here the pathway diverges to penicillins in Penicillium chrysogenum and Aspergillus nidulans and to cephalosporins and cephamycins in various molds and Actinomycetes. Cephalosporins and cephamycins (7-.alpha.-methoxycephalosporins) contain the cephem bicyclic ring system (a .beta.-lactam fused to a dihydrothiazine ring).
The enzymes catalyzing the .beta.-lactam pathway
Formation of the ACV-tripeptide is carried out by the enzyme .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS).
The enzyme catalyzing the second step in the penicillin, cephalosporin and cephamycin biosynthesis is isopencillin N synthase (IPNS or cyclase). IPNS is stimulated by ferrous ions and ascorbate, and requires a reduced environment. As the .beta.-lactam ring is formed during cyclization, isopenicillin N (IPN) is the first compound produced in the pathway with antibiotic activity.
IPNS has been purified from a wide variety of .beta.-lactam producing organisms including Streptomyces clavuligerus, Streptomyces lactamdurans, Penicillium chrysogenum and Cephalosporium acremonium.
The final step in the penicillin biosynthesis is catalyzed by acyl-CoA:6-aminopenicillanic acid transferase (AT), which has been purified from organisms as e.g. Penicillium chrysogenum and Aspergillus nidulans.
Most of the enzymes involved in the biosynthesis of .beta.-lactams have been characterized. The genes coding for ACVS, IPNS (cyclase) and AT have been cloned and modified in different ways to increase expression (Martin, J. F., J. Indust. Microorg., 9, p. 73-90, 1992).
The genes encoding the Penicillin biosynthesis
The genes of penicillin biosynthesis in P. chrysogenum and A. nidulans are named respectively pcbAB, pcbC and penDE and are tightly clustered.
The pcbAB gene, encoding the ACV synthetase (ACVS) in P. chrysogenum, is an unusually large gene of about 12 kb without any introns (Smith et al., EMBO J., 9, p. 2743-2750, 1990, Diez et al., J. Biol. Chem., 265, p. 16358-16365, 1990). Transcriptional mapping showed the presence of a long transcript of about 11.5 kb that hybridized with several probes internally in the pcbAB gene. Further two small transcripts of 1.15 kb hybridized with the pcbC or the penDE gene (Martin, J. F., J. Indu. Micr., 9, p. 73-90, 1992).
The transcriptional initiation and termination region of the pcbAB gene has been completely sequenced. The pcbAB gene is linked to the pcbC and penDE genes and is transcribed in the opposite orientation to them.
Also the pcbC gene, encoding isopenicillin N synthase or cyclase of P. chrysogenum has been sequenced. The sequence does not contain any introns and the genes of P. chrysogenum (Barredo et al., Mol. Gen. Genet, 216, p. 91-98, 1989) are very similar to those of Streptomyces griseus, N. lactamdurans, and other Actinomycetes and filamentous fungi.
The penDE gene encodes AT which is the last step of the penicillin biosynthesis (Barredo et al., Gene, 83, p. 291-300, 1989). The penDE gene of A. nidulans is very similar to the penDE gene of P. chrysogenum and contains three introns in similar positions.
Transcription of the genes of the Penicillin biosynthesis
Several groups have reported transcription analysis of the upstream regions of the pcbC and pcbAB genes. In the fungi, the divergently transcribed genes are separated by about 1 kb. Smith et al. (Bio/Technology, 8, p. 237-240, 1990) used S1 mapping and primer extension to identify transcription initiation sites in the 5' region of the C. acremonium pcbC gene. Major and minor pairs of mRNA start sites were found on either side of a pyrimidine-rich block in the promoter region at positions -64 and -72, relative to the first base of the ATG initiation codon. A consensus TATA box was observed 68 bp upstream of the first major transcription start site. A similar motif was found at position -147 in the 5' region of the A. nidulans pcbC gene.
The sequence flanking the translation initiation codon matches the consensus fungal sequence. Barredo et al. (Mol. Gen. Genet., 216, p. 91-98, 1989) mapped the start site of P. chrysogenum pcbC mRNA by primer extension and showed that a single transcript was made that originated close to the structural gene, starting at position -11.
Similar studies by Kolar et al. (J. Biotechnol., 17, p. 67-80, 1991) with a penicillin production strain of P. chrysogenum revealed two major transcription initiation sites, at -131 and -132 as well as at -397.
Primer extension studies of the A. nidulans pcbAB gene demonstrated a major mRNA start point at -230 bp. There was found no recognizable core promoter sequences, a situation frequently encountered in fungal genes. As the pcbC and pcbAB genes may be regulated in a coordinate fashion, a search was made for potential regulatory elements, such as receptor sites for transacting proteins, within the intergenic region separating the pcbAB and pcbC genes. A 53-bp region of dyad symmetry is located equidistant from the two genes, but no other extensive sequence identities were detected (McCabe et al., J. Biol. Chem., 266, p. 12646-54, 1991).
Analysis of pcbC mRNA during a C. acremonium seven-day fermentation showed a large accumulation of a 1.5-kb transcript between the second and the fourth day. This correlated with the appearance of products of the pathway after isopenicillin N (Smith et al., Bio/Technology, 8, p. 237-40, 1990). The fact that mRNA levels decreased after the fifth day when antibiotics peaked was attributed to stability of the IPNS enzyme.
Regulation of the genes of the penicillin biosynthetic pathway
Little is known about the molecular mechanisms that modify the expression of the genes that regulate the penicillin biosynthesis, despite the fact that many studies show the biosynthetic pathway is subject to numerous metabolic controls.
Recent efforts in this direction have focused on characterizing the DNA regions controlling gene expression and analysing transcription events in terms of critical cell growth parameters that affect antibiotic formation.
ACV synthesis may be the rate-limiting step in biosynthesis of penicillins and cephalosporins and is known to be regulated by glucose in Penicillium chrysogenum and Nocardia lactamdurans, by phosphate in Streptomyces clavuligerus and by ammonium in Streptomyces clavuligerus and Cephalosporium acremonium. It is also strongly affected by the oxygen transfer rate of the cultures. Regulation of pcbC expression in C. acremonium occurs primarily at the transcriptional level. Similar studies of the S. clavuligerus pcbC gene show its expression to be under transcriptional control. When cultures of S. clavuligerus were grown in rich or defined media, the amounts of pcbC mRNA correlated well with the IPNS enzyme activity and antibiotic production; in defined media, peak values of both occurred much earlier than in rich media (Y. Aharonowitz et al., Annu. Rev. Microbiol., 46, p. 461-95, 1992).
Analysis of mRNA levels of penicillin biosynthetic genes in A. nidulans, under conditions where the penicillin synthesis was repressed, showed no transcripts, suggesting common regulation of these genes at the transcriptional level (McCabe et al., EMBO J., 9, p. 279-87, 1990).
Penalva et al. (Genetics and Molecular Biology of industrial Microorganisms, Washington, D.C., Am. Soc. Microbiol., p. 256-61, 1989; Gene, 89, p. 109-15, 1990) showed that in A. nidulans the pcbC gene was transcribed only after arrest of cell growth and only then penicillin was detected in the fermentation broth.
A rather different picture was found in P. chrysogenum. Levels of pcbC mRNA and IPNS stayed about the same throughout the fermentation, both in a wild-type strain and in a highly mutated overproducer strain (Kuck et al. Appl. Microbiol. Biotechnol., 31, p. 358-65, 1989). The latter exhibited 32- to 64-fold more mRNA than the wild-type strain.
Beatriz Perez-Esterban et al. (Molecular Microbiology 9:4, p. 881-895, 1993) found that the IPNS promoter of the A. nidulans IPNS gene is mostly regulated by upstream negative control elements that act upon a high basal activity. Sequential deletion analysis of three negative cis-acting elements result in a mutated promoter that is 40 times (sucrose broth) or 12 times (lactose broth) more active than the wild type. One of these cis-acting elements is involved in sucrose repression. Strikingly, it is located outside the non-transcribed 525 bp intergenic region and maps to the coding region of the divergently transcribed pcbAB gene. A 5'-deletion up to -56 (relative to the major transcription starting point (tsp)) showed that this region contain information to provide almost half of the maximal promoter activity and allows initiation of the transcription at the correct site. By using total-protein extract from mycelia grown under penicillin producing conditions a DNA-binding activity was detected which specifically binds to a promoter fragment located between -654 and -455 (relative to IPNS tsp). Deletions covering this region partially abolish IPNS promoter activity.
The interpretation of regulatory mechanisms in mutated, high-.beta.-lactam-producing strains is complicated by possible chromosomal aberrations in the cluster of biosynthetic genes. For example, one P. chrysogenum overproducer strain had 8-10 copies of the pcbC gene (Smith et al., Mol. Gen. Genet., 216, p. 492-97, 1989) and another contained the pcbC and penDE genes in a DNA segment of at least 35 kb amplified 14-fold (Barredo et al., Curr. Genet., 16, p. 453-59, 1989). The significance of such findings is relevant for attempts to genetically manipulate high producer strains, either through introducing additional copies of .beta.-lactam biosynthetic genes to overcome pathway blocks or by altering regulatory elements.
Amplification of the pcbC-penDE gene cluster of P. chrysogenum Wis 54-1255 led to as much as a 40% improvement in production yields (Veenstra et al. J. Biotechnol., 17, p. 81-90, 1991). Increased antibiotic yields were also reported in A. nidulans transformants containing multiple copies of pcbAB and pcbC genes (McCabe et al., J. Biotechnol., 17, p. 91-97, 1991).
Attempts to increase cephalosporin C yields in C. acremonium and penicillin in P. chrysogenum by inserting multiple copies of the pcbC gene were unsuccessful (Skatrud et al. Bio/Technology, 7, p. 477-86, 1989).
A similar result was obtained in terms of penicillin production in a wild-type strain of A. nidulans (Penalva et al., Genetics and Molecular Biology of industrial Microorganisms, Washington, D.C., Am. Soc. Microbiol. p. 256-61, 1989).
Relevant patent documents
U.S. Pat. No. 4,885,251 (Eli Lilly) describes a DNA sequence from C. acremonium encoding isopenicillin N synthase (IPNS). The IPNS encoding gene sequence was isolated from C. acremonium. The intact IPNS gene (pcbC) and associated promoter has been used to construct a vector that drives the expression of IPNS in C. acremonium. Further the IPNS promoter has been fused to a hygromycin phosphotransferase-encoding DNA sequence and placed onto C. acremonium expression vector.
U.S. Pat. No. 4,892,819 (Eli Lilly) describes a DNA sequence, encoding isopenicillin N synthase (IPNS), comprising the IPNS encoding gene (pcbC) and its promoter from Penicillium chrysogenum. The DNA sequence can be placed in an expression vector that function in P. chrysogenum and C. acremonium. This can be used to increase ultimate expression of a product encoded on a recombinant DNA vector.
EP 200,425 (Eli Lilly) discloses vectors encoding isopenicillin N synthase (IPNS). The vectors permit high level expression of IPNS in C. acremonium and E. coli. The Cephalosporium vectors are useful for strain improvement, to increase efficiency and yield in fermentations for the production of penicillin and cephalosporin antibiotics. The vectors may also be modified to give vectors for increasing the production yields and efficiency of P. chrysogenum, Streptomyces clavuligerus etc. in fermentations.
EP 260,762 (Gist-Brocades) provides a transformation method for preparing Penicillium transformants. The DNA is preferably integrated into a host with stable expression of the structural gene(s) which is introduced. Particularly, complementation of auxotrophy is employed for selection.
EP 354,624 (Gist Brocades) describes a subtraction isolation method for identifying genes associated with the production of secondary metabolites in microorganisms. The method is exemplified with production of penicillin in P. chrysogenum.
EP 357,119 (Gist Brocades) discloses the clustered antibiotic biosynthetic genes encoding IPNS, AT and ACVS and are advantageously employed for improvement of production of the antibiotic in microorganisms and for the isolation of other genes involved in the biosynthesis of the antibiotic. The invention is exemplified with improved production of penicillin in P. chrysogenum, with the isolation of another clustered biosynthetic gene(s) and with the expression of clustered penicillin biosynthetic genes in Acremonium chrysogenum.
EP 448,180 (Gist Brocades) describes a method for modulating production of secondary metabolites which includes modulating the number and/or the size of the organelles, preferably microbodies, in host organism. This is done by altering the expression of a protein present in said organelles; and/or interfering with the cellular control mechanisms for maturation or fission of said organelles; and/or contacting the microorganism with agents capable of regulating the number and/or size of organelles; or modulating the cellular localization of at least one protein, optionally derived from another microorganism, directly or indirectly involved in the production of said secondary metabolites by adding, deleting or altering one or more DNA sequences encoding one or more targeting signals in the gene(s) of one or more of said proteins.